JP6998763B2 - Soluble antibody complex for activating and increasing T cells or NK cells - Google Patents

Description

Related Applications This application claims priority to US Patent Application No. 62 / 045,591 filed September 4, 2014, the entire contents of which are incorporated herein by reference.

The present disclosure relates to methods and compositions of soluble monospecific antibody complexes for activating and increasing human T or NK cells.

Background Host tolerance and adaptive immunity are complex and critical components of human health. T lymphocytes are regulatory T cells that play a major role in maintaining host resistance; various subsets, including effector subsets such as CD4 + helper T cells and CD8 + cytotoxic T cells that respond immune to environmental stimuli. It is composed of.

Methods for T cell growth and proliferation in vitro have been based on many different approaches. In certain situations, T cells are activated and increased by using accessory cells and extrinsic growth factors such as antigen presenting cells and IL-2. This requires the presence and replacement of accessory cells and growth factors in the process of T cell activation and / or proliferation.

Alternatively, the reagents used for T cell activation and proliferation consist of a combination of direct or indirect anti-CD3 antibody immobilization on a solid phase surface such as a plate or on magnetic beads. In addition to the T cell primary activation signal provided by the immobilized anti-CD3 antibody, a secondary co-stimulation signal provided by the anti-CD28 antibody is required. Exogenous growth factors or cytokines such as IL-2 can also be added to enhance T cell proliferation.

Antibodies to CD3 are important components in many polyclonal T cell stimulation protocols. For the first time, Dixon et al. Showed that immobilized anti-CD3 can mediate activation and proliferation of human T cells in the absence of cognate antigen recognition by the T cell receptor. Anti-CD3 initiates activation and proliferation signaling cascades by cross-linking components of the T cell receptor complex on the surface of T cells; therefore, immobilization is required. Subsequently, sufficient activation of T cells requires a second signal from either immobilized or soluble anti-CD28 stimulation in combination with immobilized anti-CD3, according to Baroja et al. Shown. Additional co-stimulation signals provided by adhesive ligands such as CD2, LFA-1, and other TNF family molecules such as CD137 (4-1BB) provide additional proliferation or survival signals to T cells. It is possible (Smith-Garvin et.al.).

Products for T cell activation using tissue culture plates coated with immobilized anti-CD3 antibody are available from Corning (BioCoat ™ T Cell Activation Plate, Cat # 354725) and are available to those of skill in the art. Widely prepared by researchers using known standard methods. Soluble anti-CD28 antibodies can be added extrinsically to provide the co-stimulation signals required to initiate T cell activation and proliferation (Kruisbeek et. Al.).

US Pat. No. 6,352,694 describes a method for activating and increasing T cells using anti-CD3 and anti-CD28 antibodies immobilized on magnetic particles with a diameter of 4.5 um.

U.S. Pat. No. 8,012,750B2 describes a biodegradable device for activating T cells. As previously disclosed, the method uses biodegradable microspheres indirectly coated with an antibody capable of binding and activating T cells.

U.S. Patent Application No. 14 / 035,089 describes the use of an anti-CD3 and / or anti-CD28-immobilized flexible nanomatrix to signal activation to T cells. It discloses a dextran matrix with a size of 1-500 nm that acts as a flexible scaffold that can form on the surface of T cells, on which T cell activating antibodies are immobilized.

Unlike immobilized antibodies, soluble antibody complexes are capable of giving T cells a milder activation signal. In US Patent Application Publication No. 2007/0036783, a soluble bispecific tetramer composed of a complex of a second anti-CD28 antibody with an anti-CD3 antibody that can initiate T cell activation and proliferation. The use of antibody complexes (TACs) will be described. They argue that this approach provides a milder stimulus, resulting in reduced activation-induced cell death compared to the immobilized antibody method. The inventors of this application have not fully shown that the tetrameric antibody complex is in fact soluble and not adsorbed on culture plastic products during the stimulation process. Furthermore, the inventors specifically state that the use of bispecific tetrameric antibody complexes is involved in the activation and increase of stimulated T cells.

Overview We have developed a method using a soluble monospecific tetrameric antibody complex to activate and increase human primary T cells or NK cells in vitro. In particular, the present inventors have found that the use of the soluble monospecific tetrameric antibody complex provides greater T cell activation than the use of the bispecific tetrameric antibody complex. Revealed. We also use the soluble unispecific tetrameric antibody complex with the activity of NK cells as compared to NK cells cultured without the soluble unispecific tetrameric antibody complex. It was also clarified that the conversion will be promoted.

Soluble monospecific TACs do not require the removal of large magnetic particles after cell growth and can wash cells to remove any unbound soluble TAC complex; also a plate or matrix coated with a specific antibody. There is an advantage over the immobilized antibody method because it is not necessary.

Accordingly, the present disclosure is a method of activating T cells or NK cells, comprising culturing a sample containing T cells or NK cells with a composition comprising at least one soluble unispecific complex. Each soluble unispecific complex relates to a method comprising two linked binding proteins that bind to the same antigen on T or NK cells.

In some embodiments, the composition comprises at least two different unispecific antibody complexes, one unispecific antibody complex binding to a first antigen on a T cell or NK cell and the other singular. The monospecific antibody complex binds to a second antigen on T or NK cells. Optionally, the composition comprises at least three different unispecific antibody complexes, the first unispecific antibody complex binding to the first antigen on T cells or NK cells, and the second. The second unispecific antibody complex binds to the antigen of the above, and the third unispecific antibody complex binds to the third antigen.

In some embodiments, the binding protein described herein that binds to an antigen on a T cell or NK cell is an antibody or fragment thereof. In some embodiments, the soluble monospecific complex is a tetrameric antibody complex (TAC). In some embodiments, the TAC consists of two antibodies from one animal species bound to two antibody molecules from a second species that bind to the Fc site of the antibody of the first animal species.

In certain embodiments, the methods described herein are for activating T cells. In some embodiments, the method comprises one unispecific antibody complex that binds to a first antigen on T cells and another unispecific antibody complex that binds to a second antigen on T cells. Including the step of culturing a sample containing T cells together with the composition containing. In some embodiments, the first antigen is selected from CD3, CD28, CD2, CD7, CD11a, CD26, CD27, CD30L, CD40L, OX-40, ICOS, GITR, CD137, and HLA-DR, and the second antigen. Is a different antigen selected from CD3, CD28, CD2, CD7, CD11a, CD26, CD27, CD30L, CD40L, OX-40, ICOS, GITR, CD137, and HLA-DR. In some embodiments, the first antigen is CD3. In some embodiments, the second antigen is CD28. In some embodiments, the third antigen is CD2.

In some embodiments, T cell activation is enhanced T cell proliferation, enhanced cytokine production, and / or enhanced T cell expression.

In certain embodiments, the present disclosure comprises a soluble monospecific tetramer antibody complex that targets human CD3, CD28, and CD2 in order to induce optimal polyclonal activation and proliferation of human T cells in vitro. Explain to use. In such an embodiment, the composition comprises three different soluble monospecific complexes targeting CD3, CD28, and CD2.

In another aspect, the method described herein is for activating NK cells. In some embodiments, the method comprises one unispecific antibody complex that binds to a first antigen on NK cells and another unispecific antibody complex that binds to a second antigen on NK cells. Includes the step of culturing a sample containing NK cells with the composition comprising. In some embodiments, the first antigen is selected from CD335, CD2, NKG2D, NKp44, NKp30, CD16, LFA-1, and CD27, and the second antigen is CD335, CD2, NKG2D, NKp44, NKp30, CD16, LFA. -1, and different antigens selected from CD27. In some embodiments, the first antigen is CD335. In some embodiments, the second antigen is CD2.

In some embodiments, NK cell activation is enhanced NK cell proliferation, enhanced cytokine production, and / or enhanced NK cell expression.

In certain embodiments, the present disclosure describes the use of a soluble monospecific tetramer antibody complex targeting human CD335 and CD2 to induce in vitro activation and proliferation of human NK cells. In such an embodiment, the composition comprises two different soluble monospecific complexes targeting CD335 and CD2.

The basic features and various aspects of the present invention are listed below.
[1]
A method of activating T cells or NK cells, comprising culturing a sample containing T cells or NK cells, with a composition comprising at least one soluble monospecific complex, each soluble single. A method comprising a specific complex comprising two linked binding proteins that bind to the same antigen on T or NK cells.
[2]
The composition comprises at least two different unispecific antibody complexes, one unispecific antibody complex binding to a first antigen on a T cell or NK cell and the other unispecific antibody. The method of [1], wherein the complex binds to a second antigen on T or NK cells.
[3]
The composition comprises at least three different unispecific antibody complexes, the first unispecific antibody complex binds to the first antigen and to the second antigen on T cells or NK cells. The method of [1] or [2], wherein the second unispecific antibody complex binds and the third unispecific antibody complex binds to the third antigen.
[4]
The method of any of [1] to [3], wherein the binding protein is an antibody or a fragment thereof.
[5]
The method according to any one of [1] to [4], wherein the soluble monospecific complex is a tetrameric antibody complex.
[6]
A tetramer antibody complex (TAC) is a combination of two antibodies derived from one species and two antibody molecules derived from the second species that bind to the Fc site of the antibody of the first animal species. The method of [5] consisting of.
[7]
One unispecific antibody complex binds to the first antigen on T cells and the other unispecific antibody complex binds to the second antigen on T cells, activating T cells. One of the methods [2] to [6] for this purpose.
[8]
The method of [7], wherein the first antigen is CD3.
[9]
The method of [7] or [8], wherein the second antigen is CD28.
[10]
The method according to any one of [7] to [9], wherein the third antigen is CD2.
[11]
The method of any of [1] to [10], wherein the activation of T cells is enhanced T cell proliferation.
[12]
The method of any of [1] to [10], wherein activation of T cells is enhanced cytokine production.
[13]
The method according to any one of [1] to [10], wherein the activation of T cells is enhanced T cell increase.
[14]
One unispecific antibody complex binds to the first antigen on NK cells and the other unispecific antibody complex binds to the second antigen on NK cells, activating NK cells. One of the methods [2] to [6] for this purpose.
[15]
The method of [14], wherein the first antigen is CD335.
[16]
The method of [14] or [15], wherein the second antigen is CD2.
[17]
The method of any of [1]-[6] or [14]-[16], wherein the activation of NK cells is enhanced NK cell proliferation.
[18]
The method of either [1]-[6] or [14]-[16], wherein activation of NK cells is enhanced cytokine production.
[19]
The method of any of [1] to [10], wherein the activation of NK cells is an enhanced increase in NK cells.
[20]
The method of any of [1]-[19], wherein the T cells or NK cells are of human origin.
Other features and advantages of the present disclosure will be apparent from the detailed description below. However, although the detailed description and specific embodiments show the preferred embodiments of the present disclosure, they are provided only by way of illustration and therefore, from this detailed description, various within the spirit and scope of the present disclosure. It should be understood that changes and modifications will be apparent to those skilled in the art.

Humans after 7 days of culture using a mixture of monospecific TACs specific for CD3 or CD28 or a mixture of bispecific TACs for CD3 and CD28 as assessed by the CFSE dye dilution method. The result of T cell proliferation is shown. Results of T cell activation and proliferation using soluble, monospecific TAC compositions and Dynabead® Human T-Activator CD3 / CD28 beads using untreated or blocked tissue culture plates are shown. .. The results of a short-term intracellular cytokine production assay after stimulation with a soluble monospecific TAC composition or Dynabead® Human T-Activator CD3 / CD28 beads are shown. Representative images of soluble monospecific CD3, CD28, and T cells increased with CD2 TAC or Dynabead® Human T-Activator CD3 / CD28 beads for 21 days are shown. The results of a 21-day T cell increase using either soluble monospecific CD3, CD28, and CD2 TAC or Dynabead® Human T-Activator CD3 / CD28 beads are shown. FIG. 6A shows the purity of NK cells maintained unstimulated or stimulated by monospecific CD335 / CD2 TAC in ImmunoCult-XF medium supplemented with IL-2. FIG. 6B shows changes in the total number of cells unstimulated or stimulated by unispecific CD335 / CD2 TAC. NK cells after stimulation with NKp46 (CD335) and CD2 TAC cocktails in the presence of 500 IU / mL IL-2 compared to culture of NK cells alone in the presence of 500 IU / mL IL-2. Enhancement of activation was observed.

Detailed Description The present disclosure provides a method of activating and increasing human T cells or natural killer (NK) cells in vitro using a monospecific complex such as a tetrameric antibody complex.

Accordingly, in certain embodiments, the present disclosure is a method of activating T cells, comprising the step of culturing a sample containing T cells with a composition comprising at least one soluble monospecific complex. A soluble monospecific complex provides a method comprising two linked binding proteins that bind to the same antigen on T cells. In certain embodiments, the present disclosure is also a method of activating NK cells, comprising culturing a sample containing NK cells with a composition comprising at least one soluble monospecific complex, each soluble. Also provided is a method comprising two linked binding proteins in which a monospecific complex binds to the same antigen on NK cells. In some embodiments, NK cells are cultured in the presence of one or more other cytokines, such as IL-2 and / or IL-7 or IL-15. In some embodiments, T cells or NK cells are human cells.

As used herein, the term "soluble monospecific complex" means a complex comprising two binding proteins that are directly or indirectly linked to each other that bind to the same antigen. The two binding proteins are soluble and are not immobilized on the surface, particles or beads. In some embodiments, the binding protein binds to the same antigen on T cells. In other embodiments, the binding protein binds to the same antigen on NK cells.

In some embodiments, the two binding proteins are the same binding protein and bind to the same epitope on the antigen.

As used herein, the term "bispecific complex" refers to two different bindings, either directly or indirectly linked to each other, where each binding protein binds to a different antigen on a T cell or NK cell. Means a complex containing proteins.

The term "activating T cells" includes, but is not limited to, inducing T cell proliferation, inducing cytokine production from T cells, and inducing T cell proliferation.

"Antigens on T cells" include, but are not limited to, CD3, CD28, CD2, CD7, CD11a, CD26, CD27, CD30L, CD40L, OX-40, ICOS, GITR, CD137, and HLA-DR. , Can be any antigen that activates T cells.

The term "activating NK cells" includes, but is not limited to, inducing proliferation of NK cells, inducing cytokine production from NK cells, and inducing an increase in NK cells.

"Antigens on NK cells" can be any antigen that activates NK cells, including but not limited to CD335, CD2, NKG2D, NKp44, NKp30, CD16, LFA-1 and CD27.

In certain embodiments, the binding protein is an antibody or fragment thereof. Antibody fragments that can be used include Fab, Fab', F (ab') ₂ , scFv, and dsFv fragments derived from recombinant sources and / or made in transgenic animal individuals. The antibody or fragment can be derived from any species, including mice, rats, rabbits, hamsters, and humans. Chimeric antibody derivatives, ie antibody molecules that combine variable regions of non-human animals with constant regions of humans, are also contemplated within the scope of the invention. The chimeric antibody molecule may include, for example, a humanized antibody comprising an antigen binding domain derived from an antibody of mouse, rat, or other species, along with a human constant region. Conventional methods can be used to make chimeric antibodies (eg, Morrison et al .; Takeda et al., Cabilly et al., US Pat. No. 4,816,567; Boss et al., US Pat. No. 4,816,397). See Tanaguchi et al., European Patent No. 171496; European Patent Gazette No. 0173494, British Patent No. GB2177096B). The preparation of humanized antibodies is described in EP-B 10 239 400. Humanized antibodies can also be produced commercially (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.). Chimeric antibodies are expected to be less immunogenic in human subjects than the corresponding non-chimeric antibodies. Humanized antibodies can be further stabilized, for example, as described in WO 00/61635.

Antibodies that bind to T cell antigens or NK cell antigens or fragments thereof are available as commercial products or can be prepared by those skilled in the art.

In some embodiments, two antibodies or fragments thereof that bind to the same antigen are directly linked. Direct antibody ligation can be prepared by chemically binding one antibody to another, for example by using N-succinimidyl-3- (2-pyridyldithio) propionic acid (SPDP). ..

In another embodiment, the two antibodies are indirectly linked in a soluble monospecific complex. "Indirectly linked" means that the two antibodies are not covalently directly linked to each other, but are attached via a linking moiety, such as an immune complex. In a preferred embodiment, the two antibodies are indirectly linked by preparing a tetrameric antibody complex. The tetramer antibody complex is a monoclonal antibody from the same animal species that binds to the same antigen, targeting the Fc fragment of the antibody of the first animal species, in an approximately equal molar amount of the monoclonal antibody of the second animal species. Can be prepared by mixing with. The first and second antibodies are reacted with approximately equimolar amounts of the F (ab') 2 fragment of the monoclonal antibody of the second animal species that targets the Fc fragment of the antibody of the first animal species. (See Lansdorp's US Pat. No. 4,868,109, which is incorporated herein by reference, for a description of the tetrameric antibody complex and how to prepare it).

In some embodiments, the composition comprises at least two different monospecific complexes, each binding to a different antigen on a T cell. In some embodiments, the composition comprises at least two different soluble monospecific complexes, each of which at least two different soluble monospecific complexes are CD3, CD28, CD2, CD7, CD11a, CD26, CD27, It binds to different antigens selected from the group consisting of CD30L, CD40L, OX-40, ICOS, GITR, CD137, and HLA-DR.

In certain embodiments, one unispecific complex binds to CD3 and a second unispecific complex binds to CD28.

In another embodiment, the composition comprises at least three different soluble monospecific complexes, each of which binds to one of three different antigens on a T cell. In such an embodiment, neither two monospecific complexes bind to the same antigen.

In certain embodiments, the composition consists of three different soluble monospecific complexes, one specific for CD3, the second specific for CD28, and the third specific for CD2. including.

In certain embodiments, activation of a T cell in the presence of a soluble monospecific complex comprises two different binding proteins or antibodies, each of which binds to a different antigen on the T cell. Exceeds T cell activation with sex complexes.

Samples containing T cells include, but are not limited to, whole blood, apheresis samples containing T cells or peripheral blood mononuclear cells, purified primary human T cells, or immortalized human T cell lines. Any sample for which activation is desired may be used.

In some embodiments, the composition comprises at least two different monospecific complexes, each binding to a different antigen on NK cells.

In certain embodiments, one unispecific complex binds to CD335 and a second unispecific complex binds to CD2.

In another embodiment, the composition comprises at least two different soluble monospecific complexes, each binding to one of two different antigens on NK cells. In such an embodiment, neither two monospecific complexes bind to the same antigen. In some embodiments, the composition comprises at least two different soluble monospecific complexes, each of which at least two different soluble monospecific complexes are CD335, CD2, NKG2D, NKp44, NKp30, CD16, LFA-. It binds to different antigens selected from the group consisting of 1 and CD27.

In certain embodiments, the composition comprises two different soluble monospecific complexes, one specific for CD335 and the second specific for CD2. In some embodiments, the composition comprises at least one additional soluble monospecific complex specific for an antigen selected from NKG2D, NKp44, NKp30, CD16, LFA-1, and CD27.

In certain embodiments, activation of NK cells in the presence of the soluble unispecific complex outweighs activation of NK cells without the soluble unispecific complex.

Samples containing NK cells include, but are not limited to, whole blood, aferesis samples containing NK cells or peripheral blood mononuclear cells, purified primary human NK cells, or immortalized human NK cell lines. Any sample for which activation is desired may be used.

Composition The present disclosure comprises at least one soluble unispecific complex comprising two linked binding proteins in which the soluble unispecific complex binds to the same antigen on a T cell or NK cell. Also includes.

In another embodiment, the composition comprises one soluble monospecific complex bound to one antigen on a T cell or NK cell and a second soluble monospecific complex on a T cell or NK cell. Includes two soluble monospecific complexes that bind to different antigens.

In another embodiment, the composition comprises three soluble monospecific complexes, each complex binding to a different antigen on a T cell. In certain embodiments, the antigens are CD3, CD28, and CD2.

In another embodiment, the composition comprises two soluble monospecific complexes, each complex binding to a different antigen on NK cells. In certain embodiments, the antigens are CD335 and CD2.

Uses The present disclosure includes, but is not limited to, all uses of activated T or NK cells, including, but not limited to, their use in therapy.

The methods and compositions of the present disclosure are adoptive immunotherapy for cancer, acute or persistent pathogen infections (viral, fungal, bacterial, parasitic), regulation of vaccine efficacy, or autoimmune diseases or grafts. Used to increase T or NK cells in Exvivo for in vivo use in the treatment of any disease or condition that requires T cells or NK cells, including, but not limited to, immunosuppression against host disease. can do.

The following non-limiting examples illustrate this disclosure:

Example 1
Unispecific CD3 TAC and CD28 TAC induce more vigorous proliferation in human T cells compared to the bispecific CD3 / CD28 TAC mixture (Fig. 1). Bispecific CD3 / CD28 TAC was first prepared by mixing equal doses of anti-CD3 antibody and anti-CD28 antibody. Equal doses of rat anti-mouse IgG1 were added to form bispecific TACs. The resulting TAC mixture contains 25% monospecific anti-CD3 TAC, 25% monospecific anti-CD28 TAC, and 50% bispecific anti-CD3 / anti-CD28 TAC (four doses). For a description of the body antibody complex and how to prepare it, see Lansdorp, US Pat. No. 4,868,109, which is incorporated herein by reference).

Unispecific CD3 TAC and CD28 TAC cocktails were prepared by mixing equal doses of either anti-CD3 antibody or anti-CD28 antibody with equal doses of rat anti-mouse IgG1. The resulting unispecific TAC was mixed in a 1: 1 ratio to prepare unispecific CD3 and CD28 TAC cocktails consisting of 50% anti-CD3 TAC and 50% anti-CD28 TAC.

Human T cells were concentrated from fresh whole human blood by negative isolation using RosetteSep ™ (STEMCELL Technologies Inc., Vancouver, Canada). Concentrated CD4 + T cells were labeled with CFDA-SE at a final concentration of 1uM. After labeling, CFSE-labeled T cells were resuspended in XVIVO-15 medium (Lonza, Basel, Switzerland).

A 96-well flat-bottomed tissue culture plate was blocked overnight at 4 ° C. with phosphate buffer (PBS) containing 1% human serum albumin (HSA). CFSE-labeled cells were cultured in wells blocked with 1% HSA, both in the presence of monospecific CD3 and CD28 TAC at a final concentration of 0.5 ug / mL, or with bispecific CD3 / CD28 TAC. .. Extrinsic IL-2 was not added to the culture. Samples were cultured for 7 days in a humidified 37 ° C., 5% CO2 incubator. After 7 days of culture, samples were evaluated by flow cytometry for CFSE dye dilution (an indicator of cell proliferation) and expression of CD4 and CD8.

The results show that unispecific CD3 and CD28 TACs induce more vigorous proliferation of CD4 + T cells and CD8 + T cells compared to bispecific CD3 / CD28 TACs. Proliferation of unstimulated CD4 + T cells and CD8 + T cells was minimal, 1.2% and 1.5%, respectively. Proliferation was 54.7% in CD4 + T cells stimulated by monospecific TAC, compared to 27.4% by stimulation with bispecific TAC. Proliferation was 33.0% in CD8 + T cells stimulated by monospecific TAC, compared to 15.9% by stimulation with bispecific TAC. In this result, when evaluated by CFSE dye dilution method after 7 days of culture, T cell proliferation can be induced by monospecific TAC alone, 25% of monospecific CD3 and CD28 TAC, and 50% of two. Mixtures containing highly specific TACs have been shown to be less efficient in inducing T cell proliferation.

Example 2
Soluble monospecific CD2 TAC in combination with CD3 and CD28 TAC induces optimal activation of human T cells (Fig. 2). Unispecific TACs were prepared as described in Example 1. Anti-human CD3 was used to prepare a monospecific TAC specific for anti-CD3. Unispecific anti-CD28 TAC was prepared using anti-human CD28. Unispecific anti-CD2 TACs were prepared using anti-human CD2. CFSE-labeled purified humans in 96-well plates, either untreated or blocked with 1% HSA, with various combinations of monospecific TACs or with Dynabead® Human T-Activator CD3 / CD28 beads. T cells were cultured. By flow cytometry 3 days after culture in XVIVO-15 medium, i) anti-CD3 TAC alone, ii) anti-CD3 TAC and anti-CD28 TAC, or iii) anti-CD3 TAC, anti-T cell proliferation (CFSE dye dilution method). The combined effect of CD28 TAC and anti-CD2 TAC was evaluated. On the 6th day of culture, viable cell count was performed using the Guava ViaCount assay.

Prior to cell culture, wells of 96-well tissue culture plates were left untreated or blocked overnight at 4C with PBS containing 1% HSA. Wells were washed with PBS before culturing cells. HSA prevents the antibody complex from immobilizing on tissue culture plastic products and ensures that the stimulus is mediated by soluble TAC in suspension.

Dynabead® Human T-Activator CD3 / CD28 beads (samples # 5 and # 10) are unaffected by well pretreatment and induce high levels of proliferation of both CD4 + T cells and CD8 + T cells. do. When Dynabeads® was used to increase T cells, the total viable cell count on day 6 was similar between plate treatments. CD3 TAC alone is not sufficient to induce T cell growth (samples # 2 and # 6), while the combination of monospecific CD3 and CD28 TACs is used if the wells remain untreated. Can induce high levels of T cell proliferation (Sample # 3). When CD3 and CD28 TAC were used in wells blocked with 1% HSA (Sample # 8), T cell proliferation was significantly reduced compared to untreated wells given the same stimulus, day 6 The total number of viable cells in Japan has decreased from 1.09E6 to 2.46E5. In contrast, the combination of unispecific CD3, CD28, and CD2 TAC had minimal effect of well blocking by HSA, similar to the Dynabead® Human T-Activator CD3 / CD28 beads. Samples # 4 and # 9). Taken together, these data suggest that immobilization of unispecific CD3 and CD28 TACs on plates affects their effect on T cell activation. However, when a combination of unispecific CD3, CD28, and CD2 TACs is used to stimulate T cells, the effect occurs independently of immobilization on the plate, and the unispecific TAC is soluble. Works in the style of. Due to the immobilization of anti-CD3 and anti-CD28 antibodies on the bead surface, Dynabead® Human T-Activator CD3 / CD28 beads are also unaffected by plate pretreatment.

Example 3
Soluble monospecificity CD3, CD28, and CD2 TAC can induce cytokine production by T cells (Fig. 3). Human T cells enriched with Rosette Sep ™ were cultured for 4 hours in the presence of various combinations of soluble monospecific TAC constructs or Dynabead® Human T-Activator CD3 / CD28 beads (FIG. 3). .. Breferdin A was added to the culture to allow cytokine accumulation in the cells, and evaluation was performed by flow cytometry after the culture. Cultures were harvested and stained for cell surface markers and intracellular cytokines after fixation and permeabilization.

The results show that CD3 TAC alone does not induce any cytokine production of IL-2 or IFNγ in T cells 4 hours after stimulation (Sample # 1). The combination of CD3 and CD28 TAC induces 2.4% and 2.45% IL-2 production in CD4 + T cells and CD8 + T cells, respectively (Sample # 2). CD3 and CD28 TAC also induced 0.5% IFNγ production in CD4 + T cells and 1.0% in CD8 + T cells. The combination of unispecific CD3, CD28, and CD2 TACs induced higher levels of IL-2 and IFNγ production by T cells compared to those of CD3 and CD28 TACs alone (Sample # 3). .. Dynabead® Human T-Activator CD3 / CD28 beads induced similar levels of IL-2 as the combination of CD3, CD28, and CD2, but higher levels of IFNγ (Sample # 4). ). Taken together, these results show that the combination of unispecific CD3, CD28, and CD2 TACs has higher levels of cytokines from CD4 + T cells and CD8 + T cells compared to CD3 and CD28 TACs alone. Production can be induced, while Dynabead® Human T-Activator CD3 / CD28 beads have been shown to induce higher levels of IFNγ. IL-2 promotes the induction of T cell proliferation and differentiation, while IFNγ is an effector cytokine involved in the induction of antiviral immune response.

Example 4
Representative of monospecific CD3, CD28, and CD2 TAC-stimulated T cells compared to Dynabead® Human T-Activator CD3 / CD28 bead-stimulated T cells in a 21-day process. Image (10x and 20x magnified) (Fig. 4). Dynabead® Human T-Activator CD3 / CD28 with a bead-to-cell ratio of 1: 1 in the presence of 30 U / mL recombinant human IL-2 from human T cells enriched with EasySep®. Cultivated with either beads or monospecific CD3, CD28, and CD2 TAC with a final concentration of 0.5 ug / mL. Cultures were monitored for cell growth and cell concentrations were maintained at 0.5-2 × 10E6 cells / mL. T cells were restimulated on days 7 and 17 of culture with Dynabead® Human T-Activator CD3 / CD28 beads or monospecific CD3, CD28, and CD2 TACs.

Dynabead® Human T-Activator CD3 / CD28 beads, as well as cultures stimulated by unispecific CD3, CD28, and CD2 TACs, induce T cell proliferation and proliferation with similar characteristics. The 4.5um beads used in the Dynabead® Human T-Activator CD3 / CD28 beads are clearly visible in the culture. In contrast to Dynabead® Human T-Activator CD3 / CD28 beads, in TAC-stimulated cultures, cell-sized beads are downstream, such as flow cytometry, prior to any downstream functional assay. There is no need to remove the beads because they can interfere with the assay.

Experimental example 5
Long-term culture and augmentation of human T cells with Dynabead® Human T-Activator CD3 / CD28 beads compared to unispecific CD3, CD28, and CD2 TACs (Figure 5). Dynabead® Human T-Activator CD3 / CD28 beads with a bead-to-cell ratio of 1: 1 in the presence of 30 U / mL recombinant human IL-2, or a single with a final concentration of 0.5 ug / mL. 1 × 10 E6 human T cells enriched with EasySep ™ were cultured with any of the specific CD3, CD28, and CD2 TACs. Cultures were monitored for cell growth and cell concentrations were maintained at 0.5-2 x 10E6 cells / mL. Fresh medium by restimulating T cells on days 7 and 17 of culture with Dynabead® Human T-Activator CD3 / CD28 beads or monospecific CD3, CD28, and CD2 TACs. The medium was replaced regularly.

On day 0, EasySep ™ concentrated 1 × 10 E6 T cells were plated on a 24-well tissue culture plate and Dynabead® Human T-Activator CD3 / CD28 beads or unispecific CD3, CD28. , And stimulated with CD2 TAC. At the indicated time points, total cell counts and viable cell counts were measured using a hemocytometer with trypan blue. The results show that the TAC-stimulated culture resulted in an increase in total T cell count and an increase in live T cell count 21 days after culture.

Example 6
Human NK cells were isolated by negative isolation using the EasySep Human NK Cell Isolation Kit (STEMCELL Technologies, Cat # 17955). Isolated NK cells (CD45 + CD3-) in ImmunoCult-XF medium (STEMCELL Technologies, Cat # 10981) supplemented with 500 IU / mL recombinant human IL-2, serum-free and heterologous. CD56 +) was cultured at a cell density of 1 × 10 ⁶ cells / mL. NK cells were not additionally stimulated (no stimulation) or were stimulated by a monospecific CD335 / CD2 TAC complex with a final concentration of 0.5 ug / mL for each antibody. On days 3, 6, 8, 10, and 13, total viable cell numbers were counted using a Nucelo counter and flow cytometry was used to assess the purity of NK cells. Cultures were maintained at ~ 1 × 10 ^ 6 cells / mL with the addition of fresh ImmunoCult-XF medium supplemented with 500 IU / mL IL-2.

As shown in FIG. 6A, NK cells isolated using EasySep and maintained in IL-2 supplemented ImmunoCult-XF medium, day 0, with or without additional stimulation. Purity increased from 84.5% to 94% from 84.5% to the 6th day. The purity of NK cells was maintained at similar levels until day 13 of culture.

As shown in FIG. 6B, in NK cells stimulated by the monospecific CD335 / CD2 TAC complex, the cell number increased between days 8 and 10 and the CD335 / CD2 stimulated culture. The increase continued until the 13th day, when the number of NK cells increased 1.72 times.

Although the present disclosure has described what is currently considered to be a preferred embodiment, it should be understood that the present disclosure is not limited to the disclosed examples. On the contrary, the present disclosure is intended to include various modifications and equivalent embodiments within the spirit and scope of the appended claims.

All publications, patents, and patent applications are by reference in their entirety to the same extent that each publication, patent, or patent application is specifically and individually indicated to be incorporated by reference in its entirety. Incorporated herein.

Details of citations to the references referenced herein

Claims (17)

A method of activating T cells, comprising culturing a sample containing T cells with a composition comprising a soluble monospecific complex.
The soluble monospecific complex comprises two linked binding proteins that bind to the same antigen on T cells .
The composition comprises at least one soluble monospecific complex.
The at least one soluble unispecific complex is a soluble unispecific complex comprising two linked binding proteins that bind to CD3.
The method. A method of activating NK cells, comprising culturing a sample containing NK cells with a composition comprising a soluble monospecific complex.
The soluble monospecific complex comprises two linked binding proteins that bind to the same antigen on NK cells.
The composition comprises at least two soluble monospecific complexes.
The at least two soluble monospecific complexes are soluble, including a soluble monospecific complex containing two linked binding proteins that bind to CD335, and a soluble containing two linked binding proteins that bind to CD2. A unispecific complex,
The method . The composition comprises at least three different soluble unispecific complexes, the first unispecific complex binds to CD335, the second unispecific complex binds to CD2, and the first. The method of claim 2, wherein the unispecific complex of 3 binds to a third antigen on NK cells. The composition comprises at least two different soluble unispecific complexes, of which one of the at least two different soluble unispecific complexes binds to CD3 and the other one. The method of claim 1, wherein the two monospecific complexes bind to a second antigen on T cells . The composition comprises at least three different soluble monospecific complexes.
On T cells, one of the at least three different soluble unispecific complexes is the first unispecific complex that binds to CD3 , and the other one is to the second antigen. A second unispecific complex that binds, and yet another one that is different from them is a third unispecific complex that binds to a third antigen.
The method according to claim 1 . The method according to any one of claims 1 to 5 , wherein the binding protein is an antibody or a fragment thereof. The method according to any one of claims 1 to 6 , wherein the soluble monospecific complex is a tetrameric antibody complex. A tetramer antibody complex (TAC) is a combination of two antibodies derived from one species and two antibody molecules derived from the second species that bind to the Fc site of the antibody of the first animal species. The method according to claim 7 , which comprises. The method of claim 4 , wherein the second antigen is CD28. The method of claim 5 , wherein the second antigen is CD28 and the third antigen is CD2. The method of any one of claims 1 and 4-10, wherein the activation of T cells is enhanced T cell proliferation. The method of any one of claims 1 and 4-10, wherein activation of T cells is enhanced cytokine production. The method according to any one of claims 1 and 4 to 10 , wherein activation of T cells is enhanced T cell increase. The method of any one of claims 2, 3 and 6-8, wherein the activation of NK cells is enhanced NK cell proliferation. The method of any one of claims 2, 3 and 6-8, wherein activation of NK cells is enhanced cytokine production. The method according to any one of claims 2, 3 and 6 to 8 , wherein the activation of NK cells is an enhanced increase in NK cells. The method according to any one of claims 1 to 5 , wherein the T cells or NK cells are of human origin.

Images